Plant transformation generally encompasses the methodologies required and utilized for the introduction of a plant-expressible foreign gene into plant cells, such that fertile progeny plants may be obtained which stably maintain and express the foreign gene. Numerous members of the monocotyledonous and dicotyledonous classifications have been transformed. Transgenic agronomic crops, as well as fruits and vegetables, are of commercial interest. Such crops include but are not limited to maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. Despite the development of plant transformation systems for introducing plant-expressible foreign genes into plant cells, additional improvements which allow for increased transformation efficiency are desirable and provide significant advantages in overcoming operational disadvantages when transforming plants with foreign genes.
Several techniques are known for introducing foreign genetic material into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (e.g., U.S. Pat. Nos. 4,945,050 and 5,141,131). Other transformation technology includes silicon carbide or WHISKERS™ technology. See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765. Electroporation technology has also been used to transform plants. See, e.g., WO 87/06614, U.S. Pat. Nos. 5,472,869, 5,384,253, WO 92/09696, and WO 93/21335. Additionally, fusion of plant protoplasts with liposomes containing the DNA to be delivered, direct injection of the DNA, as well as other possible methods, may be employed.
Once the inserted DNA has been integrated into the plant genome, it is usually relatively stable throughout subsequent generations. The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and may be crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties, for example, the ability to control the feeding of plant pest insects.
A number of alternative techniques can also be used for inserting DNA into a host plant cell. Those techniques include, but are not limited to, transformation with T-DNA delivered by Agrobacterium tumefaciens or Agrobacterium rhizogenes as the transformation agent. Plants may be transformed using Agrobacterium technology, as described, for example, in U.S. Pat. Nos. 5,177,010, 5,104,310, European Patent Application No. 0131624B1, European Patent Application No. 120516, European Patent Application No. 159418B1, European Patent Application No. 176112, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763, 4,940,838, 4,693,976, European Patent Application No. 116718, European Patent Application No. 290799, European Patent Application No. 320500, European Patent Application No. 604662, European Patent Application No. 627752, European Patent Application No. 0267159, European Patent Application No. 0292435, U.S. Pat. Nos. 5,231,019, 5,463,174, 4,762,785, 5,004,863, and 5,159,135. The use of T-DNA-containing vectors for the transformation of plant cells has been intensively researched and sufficiently described in European Patent Application 120516; An et al. (1985, Embo J. 4:277-284), Fraley et al. (1986, Crit. Rev. Plant Sci. 4:1-46), and Lee and Gelvin (2008, Plant Physiol. 146: 325-332), and is well established in the field.
The biology of T-DNA transfer from Agrobacterium to plant cells is known. See, e.g., Gelvin (2003) Microbiol. Molec. Biol. Rev. 67:16-37; and Gelvin (2009) Plant Physiol. 150:1665-1676. At minimum, at least a T-DNA right border repeat, but often both the right border repeat and the left border repeat of the Ti or Ri plasmid will be joined as the flanking region of the genes desired to be inserted into the plant cell. The left and right T-DNA border repeats are crucial cis-acting sequences required for T-DNA transfer. Various trans-acting components are encoded within the total Agrobacterium genome. Primary amongst these are the proteins encoded by the vir genes, which are normally found as a series of operons on the Ti or Ri plasmids. Various Ti and Ri plasmids differ somewhat in the complement of vir genes, with, for example, virF not always being present. Proteins encoded by vir genes perform many different functions, including recognition and signaling of plant cell/bacteria interaction, induction of vir gene transcription, formation of a Type IV secretion channel, recognition of T-DNA border repeats, formation of T-strands, transfer of T-strands to the plant cell, import of the T-strands into the plant cell nucleus, and integration of T-strands into the plant nuclear chromosome, to name but a few. See, e.g., Tzfira and Citovsky (2006) Curr. Opin. Biotechnol. 17:147-154.
If Agrobacterium strains are used for transfoimation, the DNA to be inserted into the plant cell can be cloned into special plasmids, for example, either into an intermediate (shuttle) vector or into a binary vector. Intermediate vectors are not capable of independent replication in Agrobacterium cells, but can be manipulated and replicated in common Escherichia coli molecular cloning strains. Such intermediate vectors comprise sequences are commonly framed by the right and left T-DNA border repeat regions, that may include a selectable marker gene functional for the selection of transformed plant cells, a cloning linker, a cloning polylinker, or other sequence which can function as an introduction site for genes destined for plant cell transformation. Cloning and manipulation of genes desired to be transferred to plants can thus be easily performed by standard methodologies in E. coli, using the shuttle vector as a cloning vector. The finally manipulated shuttle vector can subsequently be introduced into Agrobacterium plant transformation strains for further work. The intermediate shuttle vector can be transferred into Agrobacterium by means of a helper plasmid (via bacterial conjugation), by electroporation, by chemically mediated direct DNA transformation, or by other known methodologies. Shuttle vectors can be integrated into the Ti or Ri plasmid or derivatives thereof by homologous recombination owing to sequences that are homologous between the Ti or Ri plasmid, or derivatives thereof, and the intermediate plasmid. This homologous recombination (i.e., plasmid integration) event thereby provides a means of stably maintaining the altered shuttle vector in Agrobacterium, with an origin of replication and other plasmid maintenance functions provided by the Ti or Ri plasmid portion of the co-integrant plasmid. The Ti or Ri plasmid also comprises the vir regions comprising vir genes necessary for the transfer of the T-DNA. The plasmid carrying the vir region is commonly a mutated Ti or Ri plasmid (helper plasmid) from which the T-DNA region, including the right and left T-DNA border repeats, have been deleted. Such pTi-derived plasmids, having functional vir genes and lacking all or substantially all of the T-region and associated elements are descriptively referred to herein as helper plasmids.
The superbinary system is a specialized example of the shuttle vector/homologous recombination system (reviewed by Komari et al. (2006) in Methods in Molecular Biology (K. Wang, ed.) No. 343: Agrobacterium Protocols (2nd Edition, Vol. 1) HUMANA PRESS Inc., Totowa, N.J., pp. 15-41; and Komori et al. (2007) Plant Physiol. 145:1155-1160). The Agrobacterium tumefaciens host strain employed with the superbinary system is LBA4404(pSB1). Strain LBA4404(pSB1) harbors two independently replicating plasmids, pAL4404 and pSB1. pAL4404 is a Ti-plasmid-derived helper plasmid which contains an intact set of vir genes (from Ti plasmid pTiACH5), but which has no T-DNA region (and thus no T-DNA left and right border repeat sequences). Plasmid pSB1 supplies an additional partial set of vir genes derived from pTiBo542; this partial vir gene set includes the virB operon and the virC operon, as well as genes virG and virD1. One example of a shuttle vector used in the superbinary system is pSB11, which contains a cloning polylinker that serves as an introduction site for genes destined for plant cell transformation, flanked by right and left T-DNA border repeat regions. Shuttle vector pSB11 is not capable of independent replication in Agrobacterium, but is stably maintained as a co-integrant plasmid when integrated into pSB1 by means of homologous recombination between common sequences present on pSB1 and pSB11. Thus, the fully modified T-DNA region introduced into LBA4404(pSB1) on a modified pSB11 vector is productively acted upon and transferred into plant cells by Vir proteins derived from two different Agrobacterium Ti plasmid sources (pTiACH5 and pTiBo542). The superbinary system has proven to be particularly useful in transformation of monocot plant species. See Hiei et al. (1994) Plant J. (6:271-282); and Ishida et al. (1996) Nat. Biotechnol. 14:745-750.
In addition to the vir genes harbored by Agrobacterium Ti plasmids, other, chromosomally borne virulence controlling genes (termed chv genes) are known to control certain aspects of the interactions of Agrobacterium cells and plant cells, and thus affect the overall plant transformation frequency (Pan et al. (1995) Molec. Microbiol. 17:259-269). Several of the chromosomally borne genes required for virulence and attachment are grouped together in a chromosomal locus spanning 29 kilobases (Matthysse et al. (2000) Biochim. Biophys. Acta 1490:208-212).
Regardless of the particular plasmid system employed, the Agrobacterium cells so transformed are used for the transformation of plant cells. Plant explants (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants may then be regenerated from the infected plant material following placement in suitable growth conditions and culture medium, which may contain antibiotics or herbicides for selection of the transformed plant cells. The plants so obtained can then be tested for the presence of the inserted DNA.
These techniques for introducing foreign genetic material into plants can be used to introduce beneficial traits into the plants. For example, billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict. Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect-resistant plants through the introduction of Bt insecticidal protein genes has revolutionized modern agriculture and heightened the importance and value of insecticidal proteins and their genes.
Several Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully developed and in many cases registered and commercialized. These include Cry1Ab, Cry1Ca, Cry1Fa, and Cry3Bb in corn, Cry1Ac and Cry2Ab in cotton, and Cry3A in potato.
The commercial products expressing Bt proteins express a single protein except in cases where the combined insecticidal spectrum of two proteins is desired (e.g., Cry1Ab and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., Cry1Ac and Cry2Ab in cotton combined to provide resistance management for tobacco budworm).
That is, some of the qualities of insect-resistant transgenic plants that have led to rapid and widespread adoption of this technology also give rise to the concern that pest populations will develop resistance to the insecticidal proteins produced by these plants. Several strategies have been suggested for preserving the utility of Bt-based insect resistance traits which include deploying proteins at a high dose in combination with a refuge, and alternation with, or co-deployment of, different toxins (McGaughey et al. 1998, Nature Biotechnol. 16:144-146).
If Bt proteins are selected for use in combination, they need to exert their insecticidal effect independently so that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). A robust assessment of cross-resistance is typically made using populations of a pest species normally sensitive to the insecticidal protein that has been selected for resistance to the insecticidal proteins. If, for example, a pest population selected for resistance to “Protein A” is sensitive to “Protein B,” we would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.
In the absence of resistant insect populations, assessments can be made based on other characteristics presumed to be related to mechanism of action and cross-resistance potential. The utility of receptor-mediated binding in identifying insecticidal proteins likely to not exhibit cross resistance has been suggested (U.S. Pat. No. 6,855,873). The key predictor of lack of cross resistance integral to this approach is that the insecticidal proteins do not compete for receptors in a sensitive insect species.
In the event that two Bt Cry toxins compete for the same receptor, then if that receptor mutates in that insect so that one of the toxins no longer binds to that receptor and thus is no longer insecticidal against the insect, it might also be the case that the insect will also be resistant to the second toxin (which competitively bound to the same receptor). However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.
Cry1Fa is useful in controlling many lepidopteran pests species including the European corn borer (ECB; Ostrinia nubilalis (Hübner)) and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis).
The Cry1Fa protein, as produced in corn plants containing event TC1507, is responsible for an industry-leading insect resistance trait for FAW control. Cry1Fa is further deployed in the HERCULEX®, SMARTSTAX™, and WIDESTRIKE™ products.
The ability to conduct (competitive or homologous) receptor binding studies using Cry1Fa protein is limited because the most common technique available for labeling proteins for detection in receptor binding assays inactivates the insecticidal activity of the Cry1Fa protein.
Cry1Ab and Cry1Fa are insecticidal proteins currently used (separately) in transgenic corn to protect plants from a variety of insect pests. A key pest of corn that these proteins provide protection from is the European corn borer (ECB). U.S. Patent Application No. 2008/0311096 relates in part to the use of Cry1Ab to control a Cry1F-resistant ECB population.
This application describes strains of Agrobacterium tumefaciens that have been modified to increase plant transformation frequency. The use of these strains provides novel plant transformation systems for the introduction of plant-expressible foreign genes into plant cells. In addition, these strains provide additional improvements which allow for increased transformation efficiency and provide significant advantages in overcoming operational disadvantage when transforming plants with foreign genes.